118373_Vol_1.qxp
8/16/04
10:43 AM
Page 195
Disease Control
EXPRESSION OF A SUNFLOWER CC-NBS-LRR RGA AND
DEFENSE-RELATED GENES DURING AN INCOMPATIBLE
INTERACTION WITH PLASMOPARA HALSTEDII
Osman Radwan, Said Mouzeyar, Jean-Stéphane Venisse, Paul Nicolas, and
Mohamed-Fouad Bouzidi, UMR 1095 INRA- Université Blaise Pascal
"Amélioration et Santé des Plantes," 24, Avenue des Landais 63177 Aubière Cedex,
France
E-mail: Said.mouzeyar@ovgv.univ-bpclermont.fr
Abstract
Downy mildew, caused by Plasmopara halstedii, is one of the main diseases causing
economic losses in cultivated sunflower. Resistance in the host is conferred by major
genes denoted Pl. The genotype 'QIR8', which contains the Pl8 locus, was used to isolate
complete DNA genomic and cDNA RGA sequences. This RGA belongs to the CC-NBSLRR class of plant resistantce genes. The level of transcript accumulation corresponding
to this RGA was monitored by RT-PCR. The results show that this RGA is induced about
three days after infection and the accumulation decreases after nine days post-infection.
The high level of transcriptional expression of this RGA corresponds to the activation of
a hypersensitive-like reaction. We examined the transcriptional changes of some defenserelated genes that occur during compatible and incompatible combinations. In general,
transcriptional activation of defense-related genes was observed in both cases. However,
the induction was faster and greater in an incompatible reaction than during
susceptibility. The NDR1 and EDS1, two components of R genes mediated signalling
pathways as well as NPR1, an essential regulatory component of systemic acquired
resistance were also tested.
Introduction
The success of pathogen infection of a host plant depends on how fast the plant
recognizes the pathogen and activates the appropriate defense reaction. If the pathogen carries
an avr (avirulence) gene whose product is specifically recognized by the product of the
corresponding R (resistance) gene in the plant, resistance mechanisms are triggered rapidly,
resulting in disease resistance. If either the avr or the R gene is absent, the pathogen is not
recognized rapidly, defense responses are activated slowly, and disease ensues (Dangl and
Jones, 2001). During incompatible plant-pathogen interactions, recognition of a potential
pathogen often results in a hypersensitive reaction (HR), a localized activation of programmed
cell death (PCD). It has recently been reported that the simultaneous production of H2O2 and
nitric acid (NO) is required to induce the PCD occurring during the HR (Concetta de Pinto et
al., 2002). Many physiological and molecular changes are associated with the HR. Alteration
in respiration rates, electrolyte leakage and oxidative cross linking of cell wall proteins have
been reported as some of the rapid physiological changes occurring during the HR. A number
of defense genes whose products may intervene in this plant response have been extensively
studied: they include enzymes of the phenolpropanoid pathway involved in the synthesis of
Proc. 16th International Sunflower Conference, Fargo, ND USA
195
118373_Vol_1.qxp
8/16/04
10:43 AM
Page 196
Disease Control
antimicrobial phytoalexins, enzymes with hydrolytic activities, toxic compounds and cell wall
proteins (Lamb et al., 1989). In infected plants, these genes are induced around the necrosis
once it has developed, i.e., late in HR. Moreover, most of them are also strongly expressed
during compatible interactions, which result in the disease of the plant, and for some of them,
during the normal development of the plant (Lamb et al. 1989). The lack of specificity of
these defense genes as well as their activation in the late stages of the HR suggest that they
may not, by themselves, account for the establishment of the complex inducible response that
constitutes the HR, but rather may accompany this reaction.
Most plant R genes that have been isolated to date encode proteins that share structural
similarities in that they have a nucleotide-binding site (NBS) and a leucine-rich repeat (LRR).
The NBS region is preceded by either a coiled-coil (CC) domain or a so-called TIR domain
that is defined by its homology to the intracellular effector domains of the Drosophila Toll
and human interleukin-1 receptors.
Downy mildew, caused by Plasmopara halstedii (Farl.) Berl. and de Toni, is one of the
main diseases causing economic losses in cultivated sunflower (Helianthus annuus L.). The
major dominant genes denoted by Pl confer resistance to this disease. At this time only two
loci have been localized on the RFLP map of Gentzbittel et al. (1999); the first locus
(Pl1/Pl2/Pl6 locus) has been localized on linkage group 1 (Mouzeyar et al., 1995; Vear et al.,
1997; Roecel-Drevet et al., 1996) where as the second locus (Pl5/Pl8) has been localized on
linkage group 6 (Bert et al. 2001; Radwan et al. 2003). The study of resistance to sunflower
downy mildew, Plasmopara halstedii, revealed that there were two types of resistance
(Mouzeyar et al., 1994). With type I resistance, the pathogenic agent is limited to the roots
and the lower part of the hypocotyl, whereas with the type II resistance, the pathogenic agent
grows throughout the whole length of the hypocotyl and may sporulate on the cotyledons.
Radwan et al. (2004) used YSQ and QIR8 lines (which showed type II resistance) to clone,
sequence and map two full-length sequences belonging to the CC-NBS-LRR class of plant
resistance genes in sunflower.
The aim of this study was to compare the transcriptional expression of this RGA and
different defense-related genes during the compatible and incompatible reactions with
Plasmopara halstedii race 300.
Materials and Methods
Sunflower Genotypes and Infection Procedure. Infection methods were developed on
the sunflower lines QIR8 (resistant) and CAY (susceptible). The infection procedure of
Plasmopara halstedii (race 300) was carried out as described by Mouzeyar et al. (1994). The
infected plants were placed, for 2 weeks, in a growth chamber at 18r1C, with a relative
humidity of 60 to 70%, a photoperiod of 16h and a light intensity of about 200 µE/m sq/s. The
light was provided by 250 watt Osram mercury vapor lamps.
DNA and RNA Manipulations. DNA was isolated using the CTAB method, as described
by Saghai Maroof et al. (1984), whereas the RNA was extracted by using the method
described by Bogorad et al. (1983) with slight modification, using only 0.5 g of the sample.
RT-PCR Procedures. Total RNA was treated twice with DNAase 1 in the presence of
Rnasine (Invitrogen, France) to remove the genomic DNA contamination. Two µg of
DNAase treated RNA was primed with an oligo (dT)12-18 and the first cDNA strand was
synthesized using 50 units of reverse transcriptase (SuperScript First-Strand Synthesis
System for RT-PCR, Invitrogen, France), 2 µl of 10X RT-buffer, 4 µl of Mgcl2 (25 mM), 1
196
Proc. 16th International Sunflower Conference, Fargo, ND USA
118373_Vol_1.qxp
8/16/04
10:43 AM
Page 197
Disease Control
µl dNTP mix (10 mM), 2 µl DTT (0.1 M) and 1 µl of Rnasine, in 20 µl final volume. The
mixture was incubated at 42C for 75 min and then the reaction was terminated at 70C for 15
min. Finally 1 µl of Rnase H was added to each tube and incubated for 20 min at 37C. A
"minus" reverse transcriptase RT-PCR reaction was used as a control to test each mRNA
sample for genomic DNA contamination. The cDNA synthesis and RT-PCR conditions were
as described above except no reverse transcriptase enzyme was added to the cDNA synthesis
reaction.
Amplification, Cloning and Sequencing of Defense Genes. Multiple sequence
alignment (CLUSTAL-X, Thompson et al., 1997) of defense genes of other species listed in
GenBank databases at the National Center for Biological Information
(www.ncbi.nlm.nih.gov) was performed. Primers were designed within regions highly
conserved to produce RT-PCR products. This method (degenerate primers) was used for all
the defense genes used in this work except the PR5 and PDF1.2 (Xu et al., 2003), elongation
factor of Plasmopara halstedii (M.F. Bouzidi, 2004, pers. comm.) and CC-NBS-LRR
(Radwan et al., 2004), for which specific primers were used. The amplified products were
cloned into PGEM-T Easy Vector (Promega) and sequenced by Genome Express (Grenoble,
France).
RGAs and Defense Genes Expression. Upon sequence verification, specific primers
were designed. Transcriptional expression of each gene was analyzed by semiquantitative
PCR.
Results and Discussion
The predicated protein structure of the RGA clone Ha-NTIR11g was 1279 amino acids
long and showed similarity to other resistance genes. It shared 53% identity with a resistance
protein candidate (RGC 1b) from Lactuca sativa L. (Shen et al., 1998), 32% identity with an
RPP13-like protein encoded by the Arabidopsis downy mildew resistance gene from
Arabidopsis thaliana L. (Sato et al., 2000), 30% and 32% identity with an I2C-2 protein
encoded by the tomato wilt resistance gene from Lycopersicon esculentum Mill. (Ori et al.,
1997). It has 9% identity with a PU3 protein encoded by a sunflower downy mildew
resistance gene candidate from Helianthus annuus (Bouzidi et al., 2002). The amino acid
sequence predicted from Ha-NTIR11g has been divided into three domains (Figure 1). The
first is the CC domain (amino acids 1-154). This domain contains a leucine zipper (LZ)
(amino acids 26-57). The second domain is NBS (amino acids 155-542). This domain
includes P-loop (Kinase 1-a), Kinase 2 and Kinase 3-a (RNBS-B) motifs of the NBS and the
RNBS-A, RNBS-C, GLPL, RNBS-D and MHD motifs that are conserved in the NBS domain.
The third domain is the LRR and C terminus (amino acids 542-1279). Several LRR motifs
were detected in this region; however, most of them are imperfect except for three motifs.
NB
S
P Loop Kinase-2 Kinase-3a GLPL MHD
LRR
CC
Figure 1. Diagram characterization of Ha-NTIR11g cDNA. The sequence is divided into three domains: CC 'coiled
coils' domain, NBS 'nucleotide binding site' domain and LRR 'lucine rich repeat' domain. The different motifs were
identified according to Moyers et al 1999.
Proc. 16th International Sunflower Conference, Fargo, ND USA
197
118373_Vol_1.qxp
8/16/04
10:43 AM
Page 198
Disease Control
Southern hybridization analysis was carried out to detect the genomic organization of HaNTIR11g. Southern blots showed multiple bands of varying intensity in each enzyme
restriction digestion and these bands represented Ha-NTIR11g or its homologous sequences in
sunflower. Several polymorphic bands were detected between the susceptible and resistant
parents CAY and QIR8 for Ha-NTIR11g.
The level of transcript accumulation corresponding to this RGA was monitored by
semiquantitative PCR. The results show that this RGA is induced about 3 days after infection
and the accumulation decreases after 9 days post-infection. The high level of transcriptional
expression of this RGA corresponds to the activation of a hypersensitive-like reaction. We
examined the transcriptional changes of some defense-related genes that occur during
compatible and incompatible combinations. In general, transcriptional activation of defenserelated genes was observed in both cases (Figure 2).
PR
dpi
1.5
Control
3 6 9
Control
Infection
12 15 1.5 3
6
9
12 15
1.5
3
6
PS
9 12 15 1.5
Infection
3
6
9
12 15
EF1
CC
PR5
Figure 2. Semi-quantitative reverse transcription PCR. Derived amplification products were separated on TAE
agarose gels and stained with ethidium bromide. EF1= constitutive elongation factor, CC= Ha-NTIR11g RGA 'CCNBS-LRR', Control= plant uninfected, Infection= plant infected with Plasmopara halstedii race 300, PR= resistant
sunflower line (QIR8) and PS= susceptible sunflower line (CAY)
Rapid production of reactive oxygen species (ROS), some of which may be generated by
a multi-subunit NADPH oxidase complex in the plasma membrane (Doke et al., 1994), is
often associated with cell death. Rapid ion fluxes across the plasma membrane, called XR
(Atkinson et al., 1985), are also considered one of the earliest events of the HR. A lipid-based
second messenger might also be involved in the HR-signaling process. However, is still not
clear whether these signals are necessary and/or sufficient to initiate cell death. In contrast to
other defense genes, HSR203j, a tobacco gene whose activation is rapid, highly localized and
specific for incompatible plant-pathogen interaction (Pontier et al., 1994), has been shown to
be strongly correlated with programmed cell death occurring in response not only to diverse
pathogens, but also to various cell-death-triggering extracellular agents (Pontier et al., 1998).
We used degenerate primers to amplify the corresponding gene in sunflower and found that
this gene is induced about 3 days after infection, and the accumulation decreases after 9 days
post-infection in the case of an incompatible reaction. In contrast, no activation was observed
during the compatible reaction. The high level of transcriptional activation of HSR203j
coincides with the activation of a hypersensitive-like reaction as well as a high level of
transcriptional expression of the RGA.
198
Proc. 16th International Sunflower Conference, Fargo, ND USA
118373_Vol_1.qxp
8/16/04
10:43 AM
Page 199
Disease Control
References
Atkinson, M.M, Huang, J.S, and Knopp, J.A. 1985. Hypersensitivity of suspension-cultural tobacco cells to
pathogenic bacteria. Phytopathology. 75:1270-1274.
Bert, P.F, Tourvielle De Labrouhe, D., Philippon ,J., Mouzeyar, S., Jouan, I., Nicolas, P., and Vear, F, 2001.
Identification of a second linkage group carring genes controlling resistance to downy mildew (Plasmopara
halstedii) in sunflower (Helianthus annuus L.). Theor. Appl. Genet. 103:992-997.
Bogorad, L, Gubbins, E.J., Krebbers, E., Larrinua, I.M., Mulligan, B.J., Muskavitch, KMT., Orr, E.A, Rodermel.
S.R., Schantz ,R., Steinmetz, A.A., de Vos, G., and Ye, Y.K. 1983. Cloning and physical mapping of maize
plastid genes. Methods Enzymol. 97:524-55.
Bouzidi, M.F., Badaoui, S., Cambon, F., Vear, .F, Tourvielle de Labrouhe, D., Nicolas, P., and Mouzeyar, S. 2002.
Molecular analysis of a major locus for resistance to downy mildew in sunflower with specific PCR-based
markers. Theor. Appl. Genet. 104:592-600.
Concetta de Pinto, M., Tommasi, F., and De Gara, L. 2002. Changes in the antioxidant systems as part of the
signaling pathway responsible for the programmed cell death activated by nitric oxide and reactive oxygen
species in tobacco bright-yellow 2 cells. Plant Physiology. 130:698-708.
Danel, J.L, and Jones, J.D.G. 2001. Plant pathogens and integrated defense responses to infection. Nature.
411:826-833.
Doke, N., Miura, Y., Sanchez, L.M., and Kawakita, K. 1994. Involvement of superoxide in signal transduction:
responses to attack by pathogens, physical and chemical shocks, and UV radiation. In causes of photooxidative
stress and amelioration of defense in plants (Foyer CH and Mullineaux PM) London, CRS Press. p. 177-197.
Gentzbittel, L., Mestries, E., Mouzeya,r S., Mazeyrat, F., Badaui, S., Vear, F., Tourvieille de Labrouhe, D., and
Nicolas, P. 1999. A composite map of expressed sequences and phenotypic traits of the sunflower (Helianthus
annuus L.) genome. Theor. Appl. Genet. 99:218-234.
Lamb, C.J., Lawton, M.A., Dron, M., and Dixon, R.A. 1989. Signals and transduction mechanisms for activation of
plant defenses against microbial attack. Cell. 56:215-224.
Meyers, B.C., Dickerman, A.W., Michelmore, R.W., Pecherer. R.M., Sivaramakrishnan, S., Sobral, B., and Young,
N.D. 1999. Plant disease resistance genes encode members of an ancient and diverse protein family within the
nucleotide binding super family. Plant J. 20:317-332.
Mouzeyar, S., Roeckel-Drevet, P., Gentzbittel, L., Philippon, J., Tourvielle de Labrouhe, D., Vear, F., and Nicolas, P.
1995. RFLP and RAPD mapping of the sunflower PL1 locus for resistance to Plasmopara halstedii race 1.
Theor Appl Genet . 91:733-737.
Mouzeyar, S., Tourvieille De Labrouhe, D., and Vear, F. 1994. Effect of host-race combination on resistance of
sunflower (Helianthus annuus L.) to downy mildew (Plasmopara halstedii). J. Phytopathol. 141:249-258.
Pontier, D., Tronchet, M., Rogowski ,P., Lam ,E., and Roby, D. 1998. Activation of hsr203, a plant gene expressed
during incompatible plant-pathogen interactions, is correlated with programmed cell death. Mol. Plant-Microbe
Interact. 11:544-554.
Pointier, D., Godiard, L., Marco, Y., and Roby, D. 1994. HSR203J, a tobacco gene whose activation is rapid,
highly localized and specific for incompatible plant/pathogen interactions. Plant J. 5:507-521.
Radwan, O., Bouzadi, M.F., Nicolas, P., and Mouzeyar, S. 2004. Development of PCR markers for the Pl5/Pl8
locus for resistance to Plasmopara halstedii in sunflower, Helianthus annuus L. from complete CC-NBS-LRR
sequences. Theor Appl Gent. (in press).
Radwan ,O., Bouzadi, M.F., Vear, .F, Philipon, J., Tourvieille de Labrouhe, D., Nicolas, P., and Mouzeyar, S. 2003.
identification of non TIR-NBS-LRR markers linked to Pl5/Pl8 locus for resistance to downy mildew in
sunflower. Theor. Appl. Gent. 106:1438-1446.
Roeckel-Drevet, P., Gagne, G., Mouzeyar, S., Gentzbittel, L., Philippon, J., Nicolas, P., Tourvieille de Labrouhe, D.,
and Vear, F. 1996. Colocation of downy mildew (Plasmopara halstedii) resistance genes in sunflower
(Helianthus annuus L.). Euphytica. 91:225-228.
Saghai Maroof, M.A., Soliman, K.M., Jorgensen, R.A., and Allard, R.W. 1984. Ribosomal DNA spacer-length
polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proc Natl
Sci. USA. 81:8014-8018.
Sato, S., Nakmura,Y., Kaneko, T., Katoh, T., Asamizu, E., and Tabata, S. 2000. Structural analysis of Arabidopsis
thaliana chromosome 3 1. Sequence features of the regions of 4,504,864 bp covered by sixty P1 and TAC
clones. DNA Res. 7:131-135.
Shen, K.A., Meyers, B.C., Islam-Faridi, M.N., Chin, D.B., Stelly , D.M., and Michelmore, R.W. 1998. Resistance
gene candidates identified by PCR with degenerate oligonucleotide primers map clusters of resistance genes in
Proc. 16th International Sunflower Conference, Fargo, ND USA
199
118373_Vol_1.qxp
8/16/04
10:43 AM
Page 200
Disease Control
lettuce. Mol. Plant Microbe Interact. 11:815-823.
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., and Higgens, D.G. 1997. The CLUSTAL-X windows
interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res.
25: 4876-4882.
Vear ,F., Gentzbittel, L., Philippon, J., Mouzeyar, S., Mestries, E., Roeckel-Drevet ,P., Tourvieille de Labrouhe, D.,
and Nicolas, P. 1997. The genetics of resistance to five races of downy mildew (Plasmopara halstedii).
Theorz Appl. Genet. 95: 584-589.
Xu, Hu, Bideney, D.L., Yalpani ,N., Duvick, J.P., Crasta, O., Folkerts, O., and Lu, G. 2003. Overexpression of
gene encoding hydrogen peroxide-generating oxalate oxidase evokes defense responses in sunflower. Plant
Physiology. 133:170-181.
200
Proc. 16th International Sunflower Conference, Fargo, ND USA